Summary of Work: Rare DNA synthesis errors are corrected by post-replication DNA mismatch repair (MMR). Loss of MMR increases mutation rates and leads to cancer, and mutations in certain MMR genes result in infertility in model organisms. The goals of this project are to understand the biochemistry and genetics of MMR in normal eukaryotic cells, and how mutations in MMR genes lead to environmentally associated human diseases. This year we investigated the properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers that are essential for the early steps in MMR. Based on the structures of the homologous bacterial proteins, we made a number of mutants to test the importance of ATPase activity and DNA binding to MMR. We provided evidence that all four proteins have intrinsic ATPase activities and hypothesize that they operate in the order Msh6, Msh2, Mlh1 then Pms1. We investigated the contributions of various amino acids in Msh2 and Msh6 to binding to both the mismatched bases and to the DNA backbone. We demonstrated that Mlh1-Pms1 also binds to DNA, with preference for double stranded over single stranded DNA, with very high affinity and in an apparently cooperative manner. The biochemical properties were compared to genetic results indicating that ATPase and DNA binding are essential for MMR activity in vivo. These studies have important implications for the participation of these proteins in MMR, meiotic recombination and transcription-coupled excision repair. They are also important for understanding, the risk posed to individuals in the population by exposure to DNA damaging agents and the molecular basis for the initiating events in cancer and its subsequent treatment.